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Article

Rapid Biocathode Start-Up with Mixed Methanogenic–Electroactive Inocula for Enhanced Bioelectrochemical Performance

by
Tamara Joglar
,
Andrea Crespo-Barreiro
,
Mercedes Jiménez-Rosado
and
Raúl Mateos
*
Chemical, Environmental and Bioprocess Engineering Group, I4 Institute, University of León, 24071 León, Spain
*
Author to whom correspondence should be addressed.
Appl. Sci. 2025, 15(19), 10601; https://doi.org/10.3390/app151910601
Submission received: 28 August 2025 / Revised: 26 September 2025 / Accepted: 29 September 2025 / Published: 30 September 2025
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Abstract

This study explores the use of a pre-acclimated Geobacter-enriched inoculum as a novel strategy to accelerate the start-up of biocathodes. Unlike conventional inoculation with broad-spectrum communities, the proposed inoculum combines a long-term electroactive consortium, previously adapted to anaerobic bioelectrochemical conditions, with digestate produced under controlled laboratory conditions. This prior acclimation ensures the presence of Geobacter strains already conditioned to electrode-associated growth, promoting rapid colonization and early electrochemical activity. Experiments were conducted in a dual-chamber electrochemical cell equipped with a three-electrode setup polarized at −1 V vs. Ag/AgCl. The enriched biocathode reached current densities exceeding 1.4 A/m2 within 24 h, whereas the control exhibited significantly lower, less stable, and inconsistent performance. Unlike previously reported approaches based on broad-spectrum co-inoculation, this work presents a tailor-made inoculum in which the electroactive community is not only dominated by Geobacter, but also selectively preconditioned under functional bioelectrochemical conditions. This prior adaptation is a key differentiator that markedly enhances start-up efficiency. The results demonstrate that strategic enrichment with pre-acclimated Geobacter significantly accelerates start-up and improves electrochemical performance, offering a promising pathway toward more efficient and scalable bioelectrochemical systems for wastewater treatment and renewable energy generation.

1. Introduction

A bioelectrochemical system (BES) is defined as a device in which at least one of the redox reactions occurring at the electrodes is catalysed by microorganisms [1]. Similarly to other electrochemical systems, BES are reversible, thereby enabling them to operate in both galvanic and electrolytic modes. In the latter case, BES can transform electrical energy into chemical energy, generating value-added products such as methane, hydrogen and other compounds [2,3].
In contrast to conventional metal-based electrocatalysts, which often require harsh conditions, display limited selectivity, and rely on critical raw materials, microbial and enzymatic catalysts offer high substrate specificity, operation under mild and environmentally friendly conditions, and renewable origin. These advantages make biocatalysts attractive for sustainable electrochemical processes. However, they also have limitations, including low current densities, dependence on efficient electron transfer pathways, and stability issue [4]. In electrochemical biocatalysis, two main mechanisms of electron exchange have been described: mediated electron transfer (MET), which relies on soluble redox mediators but introduces overpotentials, and direct electron transfer (DET), which requires close electrical communication between the catalytic site and the electrode [5].To improve performance, strategies such as immobilization of catalysts, use of high-surface-area electrodes, and integration of enzyme or microbial cascades have been proposed. These developments contextualize the importance of advancing efficient and robust biocathode start-up procedures.
Nevertheless, the start-up process of biocathodes in bioelectrochemical systems often required several weeks to months to achieve a stable and significant current density. In many studies, as shown in Table 1, reported start-up times typically ranged from 20 to 60 days, while initial current densities could be as low as 0.01–0.1 A/m2, gradually increasing to values exceeding 1 A/m2 once the biocathode was fully developed. This prolonged start-up period not only delayed the implementation of the technology but also directly impacted the economic and operational viability of the system. Therefore, reducing the time required to achieve optimal current densities was a key objective in the development of efficient biocathodes [6].
The efficiency of the start-up process is influenced by several key factors, including the type of inoculum used [4], the materials and architecture of the electrodes, the design of the cells, the pH of the medium, the type of electrode [12], as well as the acclimation protocols employed [13,14]. In particular, the selection and conditioning of the inoculum have been shown to be critical. Previous studies have reported successful start-up acceleration when Geobacter-dominated inocula were employed [15,16]. These studies demonstrated the strong electroactive potential of Geobacter and their role in efficient extracellular electron transfer.
In this study, we focus on evaluating how the origin of the inoculum and the acclimation strategy affect biocathode performance. Specifically, we investigate whether enriching the inoculum with Geobacter can enhance the start-up dynamics. Geobacter were chosen due to their well-documented ability for direct electron transfer to cathodes and their capacity for rapid biofilm formation [12], characteristics that may promote early electrode colonization and support the establishment of methanogenic archaea.
We hypothesize that supplementing a conventional anaerobic digestate inoculum with a Geobacter-enriched medium can enhance biocathode start-up by reducing the lag phase and increasing early current generation. To evaluate this, two systems were operated in parallel: the control was inoculated only with digestate from an anaerobic digester, while the experimental system received the same digestate inoculum supplemented with medium collected from a well-performing bioanode enriched in Geobacter. This experimental design enables a direct assessment of the impact of Geobacter enrichment on the start-up, and the initial current density development.

2. Materials and Methods

2.1. Experimental Set-Up and Operation Conditions

As detailed in Figure 1, a system was designed consisting of two 500 mL H-type chambers (ADAMS & CHITTENDEN Scientific Glass, Berkeley, CA, USA) separated by a cationic membrane (CMI7000, Membranes International, Ringwood, NJ, USA). It is configured with three electrodes. The working electrode consists of pretreated carbon felt (rectangular 3 × 5 cm2, projected area 15 cm2). The pretreatment consists of an immersion in nitric acid (1 M) for 24 h followed by an immersion in acetone (1 M) for 1 h and ethanol (1 M) for 1 h each to reduce the hydrophobicity and impurities of the material [16]. A platinum mesh (Goodfellow, Huntingdon, UK) with approximately the same size as the working electrode was used as the counter electrode. An Ag/AgCl electrode 3 M KCl was used as a reference electrode, with respect to which a potential of −1 V was applied in accordance with the literature [17]. The cathode was kept under continuous stirring at 200 rpm using an IKA-WERKE RO 15 magnetic stirring plate (IKA, Königswinter, Germany), and the temperature was maintained at 30 °C using a Phytotron device (SANYO, Moriguchi, Japan).The gaseous products were collected in a 50 mL multi-layer gas sampling bag (Ritter, Schwabmünchen, Germany) made from a five-layer composite material (PET/PE/aluminium/OPA/PE with a total thickness of 131 µm). This material is chemically inert, blocks UV light and is gas-impermeable, ensuring stable sample storage. The bag is equipped with a Twist-Type Double-O-Ring valve that provides a secure, gas-tight seal. The bag was connected to the third outlet of the cathodic compartment of the H-type electrochemical reactor via flexible, gas-tight tubing (PharMed® BPT, Saint-Gobain, Avilés, Spain) and a three-way valve (PVDF, Bürkle, Baden-Wurtemberg, Germany). This setup maintained a fully closed, anaerobic system. Prior to each experiment, the entire assembly was purged with high-purity nitrogen (N2) for 15 min to displace residual air, feeding from the bottom of the liquid phase via a syringe, to ensure gas–liquid gas, and was then immediately sealed to prevent oxygen ingress. This procedure ensured the rapid establishment of anaerobic conditions in the biocathode chamber. The gas bag was periodically disconnected via the valve without exposing the system to ambient air. Gas samples were withdrawn using a gas-tight syringe (BD Plastipak, Becton Dickinson, Franklin Lakes, NJ, USA) fitted with a second three-way valve to ensure sample integrity during transfer to the gas chromatograph.

2.2. Origin of the Inoculum and Growth Media

The tailor-made inoculum was carried out using a mixture composed of three components (Figure 2): an effluent from a single chamber bioelectrochemical cell (Cell_In, 150 mL) that had been operating stably for more than three months, a fraction of optimized anaerobic digestion products (AD_In, 25 mL), and a volume of fresh medium (325 mL). This combination (30:5:65 of Cell_In:AD_In:Fresh medium) was selected based on preliminary trials, aiming to ensure a dominant presence of the electroactive, pre-conditioned consortium (Cell_In) while maintaining sufficient microbial and nutrient diversity from the anaerobic digestate (AD_In). This balance was intended to promote rapid colonization and bioelectrochemical activity while preserving system stability.
The single-chamber bioelectrochemical cell, from which Cell_In was obtained from a laboratory-scale, single-chamber, three-electrode bioelectrochemical cell, featuring a fully biological interface with both the working and counter electrodes constructed from carbon felt. Initially, the cell was inoculated with 100 mL of anaerobic digestate from the Leon municipal wastewater treatment plant, which was then mixed with 400 mL of a sterile synthetic medium containing 3.21 g/L K2HPO4, 1.57 g/L KH2PO4, 0.01 g/L CaCl2, 0.09 g/L MgCl2, 0.01 g/L MgSO4 and 0.28 g/L NH4Cl [18]. Additionally, 1 g/L each of mineral and vitamin solutions were added as described in [18]. The cell was operated in batch mode at 25 °C under potentiostatic control at −1.0 V vs. Ag/AgCl, with 7-day feeding cycles. During operation, the medium was supplemented with 0.2 g/L of both sodium acetate and sodium bicarbonate as carbon sources. Cell_In was collected directly at the end of one of these cycles and immediately transferred to the new reactor without storage, thus avoiding exposure to air. This effluent was selected due to its enrichment in electroactive microorganisms, particularly those capable of extracellular electron transfer.
The Ad_In originated from a previous semi-pilot experiment which assessed the impact of conductive fillers and bioelectrochemical systems on the anaerobic digestion of sewage sludge [19]. This inoculum consisted of stabilised digestate obtained by mixing effluents from three reactors: R1 containing PLA/carbon black fillers; R2 coupled to an external bioelectrochemical cell; and R3 acting as a control. Consequently, the Ad_In contained a diverse and well-adapted microbial consortium dominated by methanogenic archaea. Both acetoclastic (Methanosaetaceae) and hydrogenotrophic (Methanobacteriaceae) methanogens were present alongside syntrophic bacteria, including Syntrophomonadaceae and Cloacimonadales, which facilitate the degradation of fatty acids and complex organics. The inoculum was chemically characterised by a neutral pH (~7), low volatile fatty acid accumulation (<25 mg/L) and a reduced volatile solids fraction (1.1–1.6%), confirming its stability and suitability for methane production. This composition ensured that the Ad_In inoculum provided the necessary microbial activity and balanced chemical environment to promote rapid start-up and robust methanogenesis in subsequent reactors.
The remaining volume of the inoculum was supplemented with fresh medium with the composition described above [18].
For the control reactor, a 1:5 volumetric ratio mixture was used, as previously described [20], where one part corresponds to the inoculum from the anaerobic digester (AD_In) and the remaining five parts to the medium described above. Both the control and the enriched biocathode reactors were operated under potentiostatic control at −1.0 V vs. Ag/AgCl.

2.3. Extraction of DNA and Microbial Community Structure Determination

Both the microbial population of the inoculum and the biofilm at the end of the experiment were analysed and studied. The microbial communities were analysed by high throughput 16S rRNA sequencing targeting bacteria and archaea. Genomic DNA was extracted using a DNeasy PowerSoil kit (Qiagen, Hilden, Germany), followed by PCR amplification on a Mastercycler (Eppendorf, Hamburgo, Germany). PCR products were quantified using a NanoDrop 1000 spectrophotometer (Thermo Scientific, Walthham, MA, USA).
The whole DNA extract was used to sequence large 16S rRNA gene libraries, using primers 314F (5′-CCTACGGGAGGCAGCAGCAG-3′) and 518R (5′-ATTACCACCGCGGGCTGCTGGCTGG-3′) for bacterial sequences, and 349F (5′-GYGCASCAGKCGMGAAWW-3′) and 806R (5′-GGACTACVSGGGTATCTAAT-3′) for archaeal sequences. Illumina sequencing was performed by Novogene (Cambridge, UK) on a HiSeq 2500 PE250 platform, yielding an average of approximately 30,000 raw tags per sample. After quality filtering, high-quality reads were retained for downstream analysis. Bioinformatic analysis, including taxonomic assignment, was performed using QIIME (v2) and Mothur software (v1.48.0), with classification carried out against the SILVA 138 reference database.

2.4. Analytical Techniques

To ensure the correct functioning of the bioelectrochemical system, a series of parameters were monitored and measured at the end of each cycle, with a duration of 7 days. The parameters monitored included pH, measured with a BASIC 20+ meter (Crison Instruments S.A., Barcelona, Spain), and redox potential, evaluated with an IntilliCAL ORP-Redox MTC101 sensor (Hach, Loveland, CO, USA). This probe consists of a platinum measuring electrode and an internal Ag/AgCl reference electrode. The difference in potential is reported in mV and reflects the overall redox state of the culture media, with more positive values indicating oxidizing conditions and more negative values indicating reducing conditions, and dissolved oxygen concentration, determined with an HQ40d dual-channel digital multimeter (Hach, USA). These measurements were carried out following established protocols, ensuring that conditions remained within optimal ranges to facilitate microbial growth and catalysis of electrochemical reactions.
To assess cell productivity, volatile fatty acids (VFA) from C2 to C6 were quantified employing a Bruker 450-GC gas chromatograph (USA) equipped with a flame ionization detector (FID). Also, the gas bag was disconnected from the reactor, and the total gas volume (Vg) was quantified using an airtight syringe (Hamilton SampleLock, 50 mL, SampleLock Syriges, Reno, NV, USA). The gas composition, including H2, CO2, O2, N2 and CH4, was analyzed using a Varian CP3800 GC gas chromatograph (Varian Inc., Palo Alto, CA, USA) with a thermal conductivity detector (TCD), allowing the volumes of H2 and CH4 produced to be calculated and adjusted to standard temperature and pressure (STP) conditions.

2.5. Bioelectrochemical Operation and Electroanalytical Characterization

Electrochemical analyses were conducted using a Biologic VSP potentiostat (Bio-Logic Science Instruments SAS, Seyssinet-Pariset, France) with data acquisition and control facilitated by the EC-Lab software (v11.30). A chronoamperometry (CA) was performed throughout the process. The cathode was polarized at a constant electrode potential of −1 V vs. Ag/AgCl and all the generated current normalized to A/m2.
To characterize the system and evaluate the electron transfer mechanisms due to the operation and formation of a visible biofilm [21], cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) analyses were performed at several stages: at the beginning (abiotic phase) and at the end of the experiment (cycle 11). CV was performed with a potential sweep from −1 to 0.1 V against Ag/AgCl at a sweep rate of −5 mV/s. EIS measurements were carried out at the open-circuit potential (Ei = 0 V vs. Ag/AgCl) over a frequency range from 200 kHz to 100 mHz, using an AC perturbation amplitude of 10 mV. The impedance spectra were fitted to the equivalent circuit described in Section 3.4.2 using the Z-fit module of EC-Lab software (Bio-Logic).

3. Results & Discussion

3.1. Analysis of the Microbial Composition and Abundance of the Initial Inoculum

The analysis of the microbial composition and abundance of the initial inoculum is a crucial aspect of understanding the dynamics of microbial communities in various environments for that reason Figure 3. illustrates the abundance of the 20 most significant Cell_In and Ad_In bacteria used as inoculum.
The microbial community of the Cell_In was clearly dominated by more than 87% Geobacter. This inoculum contains, in smaller proportion, other interesting microorganisms such as Longilinea, a filamentous type of microbiota that can carry out acidogenesis processes using carbohydrates such as arabinose and fructose [22]. These species play a crucial role in the hydrolysis and production of organic acids. In addition, some species may establish syntrophic associations with hydrogenotrophic methanogens, taking advantage of carbohydrates to generate methane [23,24,25].
The microbial community of AD_In has a higher proportion of methanogenic archaea and bacteria associated with fermentation and anaerobic oxidation processes. Methanosaeta and Methanolinea account for a significant percentage of the population, representing approximately 21% around 6%, respectively. Methanosaeta is mainly acetotrophic, meaning that it produces methane using acetate as a substrate [26]. On the other hand, Methanolinea uses pathways fewer common for methane production [27]. Another relevant species is Syntrophomona, a genus of anaerobic bacteria that plays a crucial role in the decomposition of organic matter, especially in anaerobic environments. These bacteria are recognised for their ability to break down organic compounds in cooperation with other species, in a process known as syntrophism, where different microorganisms collaborate to break down complex substrates that a single species would not be able to metabolise efficiently [28]. Furthermore, it has been demonstrated that syntrophic bacteria in anaerobic digestion convert fatty acids, produced by acidogenic bacteria, into acetate, hydrogen, and carbon dioxide, providing essential substrates for methanogenesis [29].

3.2. Influence of Inoculum on Start-Up

Figure 4a shows how the current density evolves over time during the inoculation cycles for the biocathode enriched in Geobacter and the control. The enriched system reaches a minimum current density of approximately −1.4 A/m2 within the first 12 h, followed by a gradual recovery and stabilization above −1.2 A/m2 within the first day. In contrast, the control remains below −0.5 A/m2, with no clear trend toward stabilization. This behaviour highlights the effectiveness of the tailored inoculum.
The favourable performance of the enriched biocathode can be mainly attributed to the role of Geobacter, a genus well known for its ability to establish direct electrochemical connections with electrodes. Geobacter possesses conductive pili and outer membrane c-type cytochromes that enable direct electron transfer (DET) [30,31], which is essential for the formation of electroactive biofilms. Its cooperation with Methanosaeta further enhances this effect, as both species can form dense and conductive biofilms that stabilize current generation over time [32].
Figure 4b confirms the system’s stability over four consecutive operational cycles with fresh medium, consistently recovering to values near −2.0 A/m2 at the beginning of each cycle and subsequently stabilizing. This pattern reflects the formation of a functional and resilient electroactive biofilm. It is important to note that biocathode start-up is typically a slow and complex process [33]. In contrast, the present system achieved −1.4 A/m2 within 12 h and stabilized above −1.2 A/m2 in less than a day, outperforming typical values reported for carbon felt cathodes, ranging from −0.59 to −1.25 A/m2 [24], with few advanced systems reaching −1.60 A/m2 [18]. This performance, along with a 140% increase in current density relative to the control, demonstrates the effectiveness of the enriched inoculum and the robustness of the biocathode configuration in fostering rapid and sustained electrochemical activity.

3.3. Monitoring of Physico-Chemical Conditions During the Operation of the Enriched Biocathode

The following Figure 5 shows the monitoring of the physic-chemical parameters of the enriched biocathode. During cycles 1 to 3, the system operated under optimal conditions. Oxygen levels in the cathodic chamber remained below 0.5 mg/L, ensuring a strictly anaerobic environment suitable for the activity of methanogenic bacteria. These low-oxygen conditions promote DET mechanisms and facilitate the formation of stable electroactive biofilms [34]. Concurrently, redox potential measurements in the cathodic chamber remained in the negative range approximately 100 to 200 mV, indicating a reducing environment consistent with active microbial electron uptake [4]. pH measurements showed expected electrochemical trends: the anodic chamber became progressively acidified due to proton production during oxidation reactions, while the counter electrode compartment exhibited gradual alkalinization, as a result of proton consumption in reduction processes [35]. These opposing pH shifts confirm sustained current flow across the system. The cathodic chamber pH remained relatively stable, supporting the notion of a functional biofilm moderating local conditions. Conductivity across all chambers remained stable within a range of 6 to 8 mS/cm, reflecting consistent ion transport and electrolyte balance. This stability indicates that no significant ionic losses or concentration shifts occurred, and that the reactor maintained its electrochemical integrity [36].
From cycle 4 onward, however, a deviation was observed, marked by a substantial increase in oxygen concentration in the cathodic chamber. This shift was attributed not to microbial changes but to a mechanical failure: worn sealing joints allowed atmospheric oxygen to infiltrate the reactor. As a result, oxygen rose above 2 mg/L, disrupting the anaerobic conditions essential for the activity of Geobacter and other strict anaerobes. This oxygen intrusion was accompanied by a marked increase in redox potential, which rose sharply and stabilized around 100 mV. The oxidative shift reflects the elevated presence of oxygen and corresponds to a diminished capacity for microbial reduction processes. Nevertheless, pH gradients across the anodic and counter electrode chambers remained active, albeit with slightly reduced amplitude, indicating that electrochemical activity persisted. Importantly, conductivity remained stable throughout, suggesting that electrolyte composition and ionic conductivity were not significantly affected by the oxygen ingress. The maintained pH and conductivity profiles, despite increased oxygen and redox potential, confirm that the reactor continued to function electrochemically. This reinforces the robustness of the system and allows us to attribute performance deviations to external factors rather than failure of the inoculum or biocathode design.

3.4. Bio-Electrochemical Characterization of Biocathodes

3.4.1. Cyclic Voltammetry (CV)

The CV applied to the study system under abiotic and biotic conditions (cycle 11) is presented to evaluate the electrochemical activity in the study reactor (Figure 6). A comparison between the electrochemical responses of the biotic system of the tailored-designed and the control reactor was also carried out to determine the effectiveness of the experimental design. This comparative analysis is essential to assess whether the electroactive microbial population has been established more efficiently, showing a higher redox activity associated with its presence [2].
Figure 6a shows the CV curves obtained for the reactor under abiotic (solid line) and biotic (dashed line) conditions. Under abiotic conditions, the CV curve exhibits the characteristic form of non-faradaic behaviour, which is dominated by the capacitive charging and discharging of the electrical double layer at the electrode-electrolyte interface [37]. This suggests that the electrochemical response is primarily governed by surface charge phenomena rather than electron transfer reactions [38]. In the abiotic system, only small, broad redox features were observed, consistent with non-biological electroactive species or adsorption processes. In contrast, the biotic system exhibited anodic and cathodic peaks (at approximately −0.1 V and −0.7 V vs. Ag/AgCl, respectively), with significantly higher peak current together with a greater separation between cathodic and anodic profiles.
The presence of these peaks in the absence of biological activity indicates that they came from electroactive components in the electrolyte, adsorption/desorption processes on the electrode surface, or spontaneous electrochemical reactions, such as hydrogen evolution or CO2 reduction, which can occur under negative potentials [39,40]. Under biotic conditions, a substantial increase in current density is observed across the entire potential window, accompanied by a significant intensification of the same redox peaks. This behaviour indicates a shift towards a system with both faradaic and non-faradaic contributions, where microbial activity amplifies electrochemical processes. The increase in peak intensity suggests the involvement of bioelectrochemical reactions, which may be mediated by microbial metabolites or direct electron transfer mechanisms [8]. Furthermore, the greater separation between the oxidative and reductive sweeps in the biotic curve indicates an increase in capacitive current [41], which is likely due to the formation of an electroactive biofilm that increases the electrode’s electrochemically active surface area [39].
Figure 6b shows a comparison of the voltametric responses of two biotic systems: the Geobacter-enriched reactor and the control. The CV curve for the custom-made inoculum shows a significantly higher electrochemical response, with higher current densities and redox peaks. The increased peak intensity indicates higher faradaic activity, suggesting a more electroactive and functionally adapted microbial community [42]. Additionally, the enriched system exhibits higher double-layer capacitance, consistent with a denser and more electrochemically coupled biofilm. By contrast, the control system exhibits a flatter voltammogram with weaker redox characteristics and a lower current response, indicating that the microbial community is less electroactive or adapted to the electrochemical environment. This comparison demonstrates the effectiveness of using a Geobacter-enriched and adapted inoculum to enhance bioelectrochemical performance by promoting a microbial consortium that is better able to catalyse redox processes at the electrode interface.

3.4.2. Electrochemical Impedance Spectroscopy (EIS)

The experimental values obtained from the electrochemical impedance spectra (EIS) for both cases, the enriched biocathode with designed inoculum and the control biocathode, are presented below, together with the corresponding fit to the equivalent circuit used for characterisation [43,44].
Figure 7a shows the Nyquist diagrams for both biocathodes. The system inoculated with the enriched inoculum exhibits a distinct semicircle in the high-frequency region, which is indicative of the charge transfer process occurring between the electrode and the presence of a well-developed biofilm [45]. In the low-frequency region, a transition towards more resistive-capacitive behaviour is observed, which is associated with charge accumulation phenomena and possible transport limitations within the biofilm or the electrode’s porous structure. By contrast, the control sample does not exhibit a clearly defined semicircle, indicating less efficient charge transfer. Additionally, its low-frequency behaviour is less capacitive, suggesting lower surface charge accumulation capacity and a more heterogeneous interface.
Figure 7a also presents the fit to an equivalent circuit, with the aim of modelling and quantifying the electrochemical processes taking place in the biocathodes analysed [43,46]. The overlap between the experimental values and the fitted simulation confirms that the selected model adequately describes the response of the system, allowing to differentiate between the different phenomena involved in the electrode-biofilm interface. The proposed equivalent circuit, represented in Figure 7b, is composed of the following elements [47]:
(a)
RS: Ohmic resistance, representing the electrolyte resistance and system connections.
(b)
RCT: Charge transfer resistance between the biofilm and the bulk, represented as a semicircle in the Niquits representation [48].
(c)
Q1: The parameter Q is the coefficient associated with the Constant Phase Element (CPE), used in equivalent circuits to model non-ideal capacitive behaviour.
(d)
RBIO: Opposition to charge transfer between biofilm and electrode.
(e)
C2: which represents the capacitive behaviour of the system, associated with surface charge accumulation on the biofilm or electrode support.
The first parallel branch consists of RCT and Q, represents the non-ideal electrochemical double layer associated with the bioelectrochemical interface. The second parallel branch, consisting of RBIO and an ideal capacitor, represents the capacitive behaviour of the system associated with surface charge accumulation in the biofilm. This model enables both faradaic and non-faradaic processes to be characterised separately, which is particularly useful for biological systems with complex interfaces, such as biocathodes.
When comparing the parameters obtained from the adjustment for both biocathodes (Table 2), significant differences are observed that show the better electrochemical performance of the system inoculated with the designed inoculum. Firstly, the ohmic resistance is lower in the enriched biocathode, which indicates a better conductivity of the medium and a more efficient electrical contact [47]. Even more relevant is the difference in the charge transfer resistance, which in the enriched biocathode reaches a value of only 50.27 Ω, while in the control it amounts to 113.5 Ω. This difference reflects a higher efficiency in the electrochemical coupling between the electrode, and the microorganisms present in the biofilm [49]. This interpretation is reinforced by the value of the exponent a1 of the constant phase element, since its value is higher in the enriched biocathode, suggesting a more homogeneous and organised interface [50]. Furthermore, biofilm-associated resistance (RBIO) decreased significantly in the enriched biocathode (314.5 Ω), indicating improved electron transfer through the biofilm and confirming the formation of a more electroactive and better-adapted microbial community. This was in contrast to the control (604.9 Ω).
Conversely, the enriched biocathode exhibits ideal capacitance C2, which is higher than that of the control and indicates a greater charge accumulation capacity. Taken together, these quantitative results demonstrate that the inoculum design reduces internal system resistances and improves interface uniformity and electrochemical storage capacity. This validates the microbial enrichment strategy as an effective way to optimise biocathode performance.

3.5. Impact of Inoculum Type on Microbial Community Dynamics

The following section presents the evolution of the microbial communities in both the control and the enriched biocathodes (Figure 8). The aim is to analyse how the taxonomic composition changed over time and how these shifts correlate with the observed functional performance of each system.
The microbial community dynamics revealed marked contrasts between the enriched and control biocathodes (Figure 8). In the enriched system (Cell_In), the initial dominance of Geobacter decreased to <1% toward the end of the experiment, likely due to changes in medium conditions such as increasing dissolved oxygen and a shift of the redox potential toward more positive values [51]: Since Geobacter requires strictly anaerobic and low-potential conditions for direct electron transfer, its decline was expected under these circumstances. Nevertheless, the enriched community stabilized into a relatively homogeneous structure dominated by Paracoccus (31%), followed by Gordonia (4%) and Longilinea (>1%). This configuration coincided with the sustained production of volatile fatty acids (VFAs), specifically propionic and caproic acids (Figure 9). Although the dominant taxa are not direct VFA producers, they likely contributed by generating precursors and maintaining favorable redox conditions: Paracoccus and Gordonia can fix CO2 and generate intermediates such as acetate, ethanol, or lactate [52] while Longilinea is associated with stabilizing electron transfer within biofilms [19]. Even at <1% relative abundance, Clostridium was likely crucial for caproate synthesis via reverse β-oxidation, using acetate and ethanol as precursors [53]. Thus, VFAs detected in the enriched biocathode were plausibly produced through syntrophic cooperation, where dominant genera provided intermediates and redox balance, enabling Clostridium to perform chain elongation.
In contrast, the control biocathode (AD_In) exhibited a heterogeneous microbial community lacking clear functional dominance. Although methanogenic genera (Methanobacterium, Methanobrevibacter, Methanosaeta) were detected, each accounted for <5% of the population, consistent with the absence of methane and VFA production. This lack of a structured electroactive consortium explains the poor electrochemical stability of the control, as cooperative biofilms are typically required for efficient electron transfer [54].
The role of the tailored inoculum becomes evident when comparing initial compositions: in Cell_In, Geobacter accounted for only a minor fraction (<5%) relative to Paracoccus and Aquamicrobium, yet its pre-acclimation under bioelectrochemical conditions likely acted as a functional seed that accelerated electrode colonization and early current onset. The persistence of electrochemical activity after the decline of Geobacter highlights potential functional redundancy, whereby other taxa (e.g., Syntrophomonas, Methanolinea) may partially sustain electron transfer and redirect reducing equivalents into caproate and propionate production. Taken together, these findings demonstrate that microbial community dynamics not only determine electron transfer efficiency but also shape the distribution of end-products. A low-abundance but pre-adapted electroactive population can drive start-up, while subsequent shifts establish alternative metabolic pathways such as chain elongation.

4. Conclusions

This study demonstrates that the use of a Geobacter-enriched inoculum in the biocathode significantly improves start-up performance in bioelectrochemical systems. Compared to non-targeted inoculation strategies, this approach led to a faster and more stable start-up, as well as the rapid development of a homogeneous and electroactive biofilm. Product formation, specifically the generation of propionic and caproic acids, was observed within a short operational period, reflecting the functional integration of the microbial community. Importantly, these outcomes indicate that the main benefit of the inoculation strategy lies in accelerating electron transfer and stabilizing biofilm–electrode interactions. Bioelectrochemical analyses further confirmed this performance, showing higher capacitive behaviour and substantially lower charge transfer resistance, indicative of enhanced electron exchange between the biofilm and the electrode. Remarkably, all these results were achieved despite operational setbacks, such as the progressive loss of anaerobic conditions due to oxygen leakage. This underscores the robustness and adaptability of the enriched microbial community and highlights the effectiveness of a functionally guided inoculation strategy to reduce start-up times, improve biocathode performance, and ensure greater operational stability. Future research should focus on systematically optimizing inoculum composition and ratios to further enhance start-up efficiency and tailor microbial communities for specific bioelectrochemical applications, while also incorporating replicated experiments to strengthen the statistical robustness and reproducibility of the findings. Furthermore, it is also important to evaluate methane selectivity and yield of these systems and use continuous gas feedings to facilitate scalability. In addition, detailed electrochemical characterization (e.g., overpotential, Tafel slope, and Faradaic efficiency) will be required to disentangle whether the observed improvements are primarily associated with methane catalysis or with general electron transfer mechanisms.

Author Contributions

Conceptualization., M.J.-R. and. R.M.; methodology, T.J. and A.C.-B.; software, T.J. and A.C.-B.; validation, M.J.-R. and R.M.; formal analysis, T.J. and A.C.-B.; investigation, T.J., A.C.-B., M.J.-R. and R.M.; resources, M.J.-R. and R.M.; data curation, T.J. and A.C.-B.; writing—original draft preparation, T.J. and A.C.-B.; writing—review and editing, M.J.-R. and R.M.; supervision, M.J.-R. and R.M. project administration, R.M.; funding acquisition, R.M. All authors have read and agreed to the published version of the manuscript.

Funding

The authors acknowledge project PID2023-147848OB-I00, financed by MICIU/AEl/10.13039/501100011033 and by FEDER, EU. In addition, the authors acknowledge the research contract of T. Joglar (INVESTIGO PROGRAM, Next Generation UE).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The raw data supporting the conclusions of this article will be made available by the authors on request.

Conflicts of Interest

The authors declare no conflicts of interest.

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Applsci 15 10601 g001
Figure 1. Schematic representation of the dual-chamber reactor used for electrochemical assays.
Figure 1. Schematic representation of the dual-chamber reactor used for electrochemical assays.
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Figure 2. Inoculation procedure for the dual-chamber bioelectrochemical system.
Figure 2. Inoculation procedure for the dual-chamber bioelectrochemical system.
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Figure 3. Taxonomic classification of 16S rDNA Amplicon Sequencing of Bacteria and Archaea at genus level or the 20 most abundant species for inoculum Cell_In and AD_In. * The evidence is not sufficiently conclusive to definitively differentiate between these genera.
Figure 3. Taxonomic classification of 16S rDNA Amplicon Sequencing of Bacteria and Archaea at genus level or the 20 most abundant species for inoculum Cell_In and AD_In. * The evidence is not sufficiently conclusive to definitively differentiate between these genera.
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Figure 4. (a) Current density evolution during the electrochemical start-up period for the biocathode inoculated with a microbial community enriched in Geobacter (blue) and for the control without specific enrichment (orange). (b) Current density evolution during the first four electrochemical cycles after inoculation with fresh medium in the enriched biocathode.
Figure 4. (a) Current density evolution during the electrochemical start-up period for the biocathode inoculated with a microbial community enriched in Geobacter (blue) and for the control without specific enrichment (orange). (b) Current density evolution during the first four electrochemical cycles after inoculation with fresh medium in the enriched biocathode.
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Figure 5. (a) Evolution of pH in the working electrode (biocathode) and counter electrode chambers during successive electrochemical cycles. (b) Evolution of dissolved oxygen, conductivity, and redox potential in the biocathode chamber over the course of the experiment.
Figure 5. (a) Evolution of pH in the working electrode (biocathode) and counter electrode chambers during successive electrochemical cycles. (b) Evolution of dissolved oxygen, conductivity, and redox potential in the biocathode chamber over the course of the experiment.
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Figure 6. (a) Cyclic voltammetry (CV) of the system inoculated with a tailor-made microbial consortium under abiotic (dashed line) and biotic (solid line) conditions. (b) Cyclic voltammetry (CV) comparison between the biotic control (orange line) and the system with the tailor-made inoculum (blue line).
Figure 6. (a) Cyclic voltammetry (CV) of the system inoculated with a tailor-made microbial consortium under abiotic (dashed line) and biotic (solid line) conditions. (b) Cyclic voltammetry (CV) comparison between the biotic control (orange line) and the system with the tailor-made inoculum (blue line).
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Figure 7. (a) Nyquist plot comparing the tailor-made inoculum biocathode with the control one. (b) Equivalent electrical circuit used to fit the Nyquist plots.
Figure 7. (a) Nyquist plot comparing the tailor-made inoculum biocathode with the control one. (b) Equivalent electrical circuit used to fit the Nyquist plots.
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Figure 8. Taxonomic classification of bacterial and archaeal 16S rDNA amplicon sequencing at genus level for the 20 most abundant species (a) the control and (b) the tailor-made inoculum.
Figure 8. Taxonomic classification of bacterial and archaeal 16S rDNA amplicon sequencing at genus level for the 20 most abundant species (a) the control and (b) the tailor-made inoculum.
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Figure 9. Production of caproic and propionic acids throughout the cycles in an enriched biocathode. Different letters show significant differences in the values (p < 0.05).
Figure 9. Production of caproic and propionic acids throughout the cycles in an enriched biocathode. Different letters show significant differences in the values (p < 0.05).
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Table 1. Start-up times and current densities reported for microbial electrochemical cell biocathodes under different experimental conditions. Values vary as a function of substrate, applied potential and microbial community.
Table 1. Start-up times and current densities reported for microbial electrochemical cell biocathodes under different experimental conditions. Values vary as a function of substrate, applied potential and microbial community.
Start-Up Time (Days)Current Density (A/m2)Reference
280.2–0.8[7]
601.2[8]
430.1–0.3[9]
30-[10]
40-[11]
171.5[12]
Table 2. EIS fitting data for tailor-made inoculum and control biocathodes.
Table 2. EIS fitting data for tailor-made inoculum and control biocathodes.
Tailor-Made InoculumControl
Rs (Ω)2.127.67
RCT (Ω)50.3113.5
Q1 (mFs(a−1))135.123.6
a0.740.20
RBIO (Ω)314.5604.9
C2 (mF)22.713.5
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Joglar, T.; Crespo-Barreiro, A.; Jiménez-Rosado, M.; Mateos, R. Rapid Biocathode Start-Up with Mixed Methanogenic–Electroactive Inocula for Enhanced Bioelectrochemical Performance. Appl. Sci. 2025, 15, 10601. https://doi.org/10.3390/app151910601

AMA Style

Joglar T, Crespo-Barreiro A, Jiménez-Rosado M, Mateos R. Rapid Biocathode Start-Up with Mixed Methanogenic–Electroactive Inocula for Enhanced Bioelectrochemical Performance. Applied Sciences. 2025; 15(19):10601. https://doi.org/10.3390/app151910601

Chicago/Turabian Style

Joglar, Tamara, Andrea Crespo-Barreiro, Mercedes Jiménez-Rosado, and Raúl Mateos. 2025. "Rapid Biocathode Start-Up with Mixed Methanogenic–Electroactive Inocula for Enhanced Bioelectrochemical Performance" Applied Sciences 15, no. 19: 10601. https://doi.org/10.3390/app151910601

APA Style

Joglar, T., Crespo-Barreiro, A., Jiménez-Rosado, M., & Mateos, R. (2025). Rapid Biocathode Start-Up with Mixed Methanogenic–Electroactive Inocula for Enhanced Bioelectrochemical Performance. Applied Sciences, 15(19), 10601. https://doi.org/10.3390/app151910601

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